Sex differences in 50 kHz call subtypes emitted during tickling-induced playful behaviour in rats.

'Tickling' induces positive affective states in laboratory rats as evidenced by the production of 50-kHz ultrasonic vocalisations (USVs), although this has mostly been investigated in males. Juvenile rats emit distinctive 50-kHz USV subtypes. Frequency-modulated (FM) 50-kHz USVs are thought to be associated with positive affect and flat 50-kHz USVs with social communication. FM and flat USVs are produced by both sexes during tickling, but it is unclear whether these calls are produced in relation to particular play-related behaviours, and whether USV subtypes are used in a sexually dimorphic manner during tickling. We tested the hypotheses that FM USVs are associated with tickle-induced play behaviours in a sex-specific way, and that flat USVs are associated with non-play activities. Rats were allocated to one of two treatment groups: tickling (tickled, n = 16/sex) or no hand contact (control, n = 16/sex). Play behaviours (hopping, darting and hand approaches) and FM and flat USVs emitted during the testing session were quantified for each rat, with the frequency of FM and flat USVs made in anticipation of, and during, each behaviour analysed. In females, play behaviours were associated with more flat USVs than in males (before and during; p < 0.001), irrespective of treatment. FM USVs were paired with hopping and darting (before and during; p < 0.001), and in anticipation of hand approaches (p < 0.001) in both tickled females and males compared to controls (both sexes) suggesting that FM USVs are linked with play behaviour. The higher call rate of flat USVs paired with play behaviour in females suggests that there may be sex differences in the role of flat USVs during play. This result is evidence of sex differences in tickle-induced behaviours and has implications for our understanding of the function of different USVs in juvenile female and male rats.

Stage 1 Registered Report: Refinement of tickling protocols to improve positive animal welfare in laboratory rats.

Rat tickling is a heterospecific interaction for experimenters to mimic the interactions of rat play, where they produce 50 kHz ultrasonic vocalisations (USV), symptoms of positive affect; tickling can improve laboratory rat welfare. The standard rat tickling protocol involves gently pinning the rat in a supine position. However, individual response to this protocol varies. This suggests there is a risk that some rats may perceive tickling as only a neutral experience, while others as a positive one, depending on how tickling is performed. Based on our research experiences of the standard tickling protocol we have developed a playful handling (PH) protocol, with reduced emphasis on pinning, intended to mimic more closely the dynamic nature of play. We will test whether our PH protocol gives rise to more uniform increases in positive affect across individuals relative to protocols involving pinning. We will compare the response of juvenile male and female Wistar rats as: Control (hand remains still against the side of the test arena), P0 (PH with no pinning), P1 (PH with one pin), P4 (PH with four pins). P1 and P4 consist of a background of PH, with treatments involving administration of an increasing dosage of pinning per PH session. We hypothesise that rats exposed to handling protocols that maximise playful interactions (where pinning number per session decreases) will show an overall increase in total 50 kHz USV as an indicator of positive affect, with less variability. We will explore whether behavioural and physiological changes associated with alterations in PH experience are less variable. We propose that maximising the numbers of rats experiencing tickling as a positive experience will reduce the variation in response variables affected by tickling and increase the repeatability of research where tickling is applied either as a social enrichment or as a treatment.